Tetrahydrofolate is an essential cofactor for many biosynthetic enzymes. It acts as a carrier of one-carbon units in the syntheses of such critical metabolites as methionine, purines, glycine, pantothenate, and thymidylate. For example, the enzyme ketopantoate hydroxymethyl transferase, encoded by the panB gene, requires a tetrahydrofolate cofactor to synthesize precursors of pantothenate. As tetrahydrofolate is synthesized de novo in bacteria, inhibition of its synthesis kills cells. Indeed, two very effective antibiotics, sulfonamide and trimethoprim, kill bacterial cells by blocking tetrahydrofolate production. These two antibiotics, which are often used in combination with each other, are commonly prescribed for the treatment of urinary tract infections, enteric infections such as shigellosis, and respiratory tract infections. The success of these drugs is indicative of the vulnerability of many pathogenic bacteria to inhibitors of tetrahydrofolate synthesis.
Bacteria have a multiple step pathway for the synthesis of the tetrahydrofolate cofactor. In one branch of the pathway, the metabolites chorismate and glutamine are substrates for aminodeoxychorismate synthase, encoded by the B. subtilis genes, pabA and pabB, which produces 4-amino 4-deoxychorismate. Aminodeoxychoismate lyase, encoded by B. subtilis pabC, then converts 4-amino 4-deoxychorismate to para-aminobenzoic acid (PABA), an important precursor. In another branch, a number of enzymes, including those encoded by B. subtilis mtrA, folA, and folK, produce the precursor 2-amino-4-hydroxy-6-hydroxy methyl-7,8-dihydroxpteridine diphosphate. This precursor and PABA are substrates for dihydropteroate synthetase, encoded by the B. subtilis sul gene (homologous to the E. coli dhps and folP genes), which produces dihydropteroate. Sulfonamides, such as sulfamethoxazole, are competitive inhibitors of dihydropteroate synthase.
Dihydropteroate is modified by the bifunctional enzyme encoded by B. subtilis folC to produce dihydrofolate. Finally, DHFR (dihydrofolate reductase), encoded by B. subtilis dfrA, modifies this dihydrofolate to generate the end product tetrahydrofolate. Trimethoprim is a competitive inhibitor of bacterial DHFRs. This selectivity is critical, as eukaryotic DHFRs are unimpeded by the antibiotic.
The emergence of antibiotic resistant strains of bacteria has heightened the urgency of developing new antibiotics. Indeed, resistance to sulfonamides and trimethoprim is prevalent and spreading. For example, before 1983, trimethoprim resistance in Shigella was rare and occurred in less than 4% of cases. However, by the 1990s, the frequency of resistance in isolates was as much as 52% (Huovinen (1997) Clinical Infectious Disease 24 (Suppl. 1):S63–66). Similarly, as many as 42% of Shigella species are sulfonamide resistant (Huovinen, supra). Often resistance to these antibiotics is a plasmid borne trait that can be transmitted horizontally to other bacterial species.